DESCRIPTION: The studies in this proposal are aimed at dissecting the molecular and biochemical events that occur during meiosis. The availability of T-DNA tagged lines that exhibit alterations in the meiotic cell cycle provide an opportunity to identify novel genes specifically involved in meiosis as well as proteins that play a conserved role in both the meiotic and mitotic cell cycle. T-DNA containing lines of Arabidopsis have been screened for male sterility and approximately 50 transformed lines that exhibit reduce male fertility have been identified. Most of these mutations result in the production of coenocytic microspores with variable numbers of nuclei. These mutants have been shown to fall into three phenotypic classes. One of these classes affect chromosome segregation, but not the timing or progression of meiotic events, suggesting that they may be defective in chromosome pairing or recombination. A second class of mutations appear to be defective in microsporogenesis. A third class of mutations, which are the subject of this proposal, appear to affect the meiotic cycle. Plants homozygous for these mutations initiate meiosis, but soon lose all synchrony and exhibit number defects in meiosis I and II. Synaptonemal complexes can be detected in these mutants and they also appear to be defective in callose deposition. These observations suggest that a protein responsible for release from a meiotic checkpoint could be missing from these cells. In order to determine which aspects of meiosis are affected in these mutants and better understand the nature of the mutations, chromosome morphology and behavior and alterations in the cytoskeleton are currently being examined. The PI proposes to isolate the genes altered in two mutants (7219 and 7953) that represent this third class of mutations. Specific goals for this project are to characterize these two mutations at the molecular level and to determine the nature of the defects in these mutants at a cellular and biochemical level. Preliminary results show that the mutations cosegregate with kanamycin resistance, suggesting that they are caused by insertion of a T-DNA sequence. For the 7219 mutation, two T-DNA/plant junction fragments have been isolated, suggesting that there are two T-DNA insertions within the genome. Genomic clones corresponding to the wild type complement of each of these sequences have been isolated. In order to determine which of the two T-DNA/junction fragments (if any) correspond to the mutant gene, three lines of experiments will be pursued. Backcrossing will be used to try and separate the two inserts within the 7219 mutant. These studies will also allow the PI to map the mutation and provide information concerning the overall recombination rates in plants heterozygous for the 7219 mutation. Northern hybridization will be used to determine if open-reading frames present within the two genomic clones hybridize with RNA isolated from wild type floral buds. Any region of the genomic clones that found to hybridize with RNA in these experiments will be retested using bud RNA isolated form the mutant. If the sterility phenotype is the result of gene disruption, then differences in transcript patterns between sterile and fertile plants should be observed. Complementation studies will be used as a third approach to identify the mutant gene sequence. Wild type sequences corresponding to the two T-DNA/junction fragments will be cloned into a gentamicin-resistant vector and transformed into wild type Arabidopsis. These plants will be crossed with 7219 heterozygotes and plants that are kan resistant/gentamicin resistant will be identified and these will be tested for male sterility. Parallel studies are proposed to isolate and identify the wild type complement of the 7593 mutation. Once genomic clones corresponding to these two mutations are identified, they will be used to isolate cDNA clones which will be sequenced. RNA hybridization studies will be used to characterize the developmental regulation of these genes during plant growth in order to determine if these genes are temporally regulated. Since the 7219 mutation also appears to affect megasporogenesis, expression of this gene will also be analyzed in developing ovules. In situ hybridization studies will then be used to provide detailed information concerning the expression pattern of these genes during plant development. 7219 and 7593 wild type cDNA clones will be used to overexpress the proteins encoded by these genes for the purpose of antibody production. These antibodies will be used to characterize the 7219 and 7593 gene products using western blot and immunolocalization studies. Epitope-tagged versions of the two cDNAs will also be reintroduced into plants as a method for localizing the cellular location of these proteins.